Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Contributions of the beta-barrel domain in the assembly of autotransporter Ag43 (#249)

Xiaojun Yuan 1 , Fiona Lewis 1 , Alvin W Lo 2 , Mark A Schembri 2 , Denisse L Leyton 1
  1. Australian National University, Canberra, ACT, Australia
  2. The University of Queensland, Brisbane, Queensland, Australia

Autotransporters are a large family of outer membrane proteins that play key roles in the

pathogenesis of many infectious diseases. Their virulence function is dependent on their

correct assembly at the bacterial cell surface. This requires a C-terminal b-barrel domain to

be folded and inserted into the outer membrane to facilitate translocation of the N-terminal

passenger domain to the bacterial cell surface where it exerts its virulence function. The

autotransporter of interest, antigen 43 (Ag43) is found in uropathogenic Escherichia coli and

mediates biofilm formation by cell-cell autoaggregation after being processed from its

cognate b-barrel only to remain attached to this domain in a non-covalent manner. To date,

the majority of Ag43 research has focused on the passenger domain, leaving the structure and

function of the Ag43 b-barrel domain uncharacterized. Using biochemical and biophysical

methods, the current study showed the fifth extracellular loop of the Ag43 b-barrel domain,

which connects b-strands 9 and 10, is required for the correct folding and function of the

Ag43 passenger domain. We also developed an in vitro refolding assay for Ag43. The results

are the first to ever provide evidence that processing of the Ag43 passenger domain occurs

via an autocatalytic cleavage mechanism. These findings provide valuable insights into the

assembly pathway of Ag43 and reveal new potential therapeutic targets in treating UPEC